The present application relates to a device that allows for handling, storage, reception, and treatments of samples in as universal manner as possible, i.e., for the reception, for different treatments, and whereby the samples are handled in μl volumes.
Essential components of biological samples, biomolecules, analytes, and essential analytes are terms that are used within this invention as synonyms, particularly for such components to in the samples to be examined that are in the focus of the examiner's or user's interest and are contained together with the matrix. These are, for example, metabolites, nucleic acids, peptides, proteins, particles, cells, viruses, microorganisms, spores, markers, enzyme substrates or products, and others as a whole or parts thereof and/or complexes from them. Within this application, the term matrix stands for such components of a sample that are not in the focus of interest and often disturb the analysis processes, particularly the solvent and other accompanying substances, for example, salts, detergents, buffers, metal ions, antibiotics, and others.
The essential components of a sample usually differ from the matrix components in their size or molecular size.
In the course of preparative and analytical procedures, biological samples must be variously treated with respect to their essential components.
Such treatments include the addition of auxiliary reagents, such as urea or guanidine for denaturation, mercaptoethanol or dithiothreitol for reduction, iodoacetamide for modification, enzymes and their cofactors for the selective elimination of modifications or the selective digestion of essential analytes, substrates for proof of enzymatic activities, inhibitors for the suppression of undesired enzymatic reactions, capture reagents for binding undesirable substances, among others.
Frequently, it is necessary to transfer the essential components of biological samples into another medium, another matrix (see above), to ensure the compatibility in subsequent cleaning processes or for analytical procedures. As often only small quantities of possibly very precious samples are available and several analytical processes may be used for one sample it is recommended to transfer only very few partial quantities of the sample into the corresponding medium that is compatible with the relevant analysis. For mass spectrometry analyses a desalting is additionally required, i.e. a very drastic reduction of salt and detergent concentration in the matrix resulting in the receipt of low-molecular essential components in the range from 1000 to 3000 Da.
Although mass spectrometry procedures can be carried out with very small volumes (1-5 μl) they require relatively high analyte concentrations. To reach the sensitivity range it is often necessary to strongly concentrate the essential components of the samples in a preanalytical process. However when doing this, the concentrations of disturbing accompanying substances must not be increased in the same extent.
Analytical screening procedures and proteomics-based technologies cope with very large numbers of samples of the same type with each of said samples is to be submitted to a treatment or several identical treatments, such as medium changes, desalting or concentration procedures.
Normally, the medium change, which can also include a desalting process, is performed by three alternatively usable methods: gel chromatography, reverse-phase chromatography, and dialysis.
The gel chromatography is based on the chromatographic separation of matrix components in the dependency of the molecular weight. This process requires clean chromatographic conditions and at present relatively large sample volumes. The sample components after the medium change are always obtained in larger volumes than the source material and nowadays they are mostly in the ml-range. So far, the experts do not know a solution for a parallelization, i.e. the simultaneous handling of a plurality of samples of the same kind. A miniaturization would be imaginable but it requires sample-volume-dependent minimum separation lengths of the mini columns and thus also the volumes of the desalted samples that do not fall under a minimum value. In WO 9502182, for example, such a procedure is described in miniaturized scale for the prefractionation for capillary electrophoresis procedures.
Compared with this, parallelized analytical chromatographic separations to the nanofluidic scale are possible (e.g. WO 2004101151). However, this method cannot be used for a preparative application during sample pretreatment because the sample quantities fall below the sensitivity range of, for example, mass spectrometry procedures, and the sample components after chromatography are always obtained in larger volumes, i.e. are additionally diluted compared to the source sample.
By means of reverse-phase chromatography preferably hydrophobic sample components are bound to a solid hydrophobic phase. Afterwards, these supporting materials are washed, and during this procedure a potential loss of essential, particularly hydrophobic analytes, is possible, and then they are eluted in the desired salt-free medium. Here, concentration steps will be feasible, if advantageous adsorption and elution volumes are selected. Microprocedures have been developed for sample and elution volumes in the μl range in ZipTips of the company Millipore (WO 9837949) or in PerfectPure tips (company Eppendorf). A parallelization of these microprocedures is difficult to carry out or cannot be carried out at all due to the running behavior of the microcolumns, membranes or monolithic structures integrated into the pipette tips that is to be controlled by the analyzer.
Parallelizable spin-down systems that are arranged in arrays of hydrophobic membranes exist. Although a centrifuge must be used, these methods approximately correspond to a feasible parallelized handling (U.S. Pat. No. 6,998,047). However, these systems require considerably larger volumes than zip tips.
The solid phase extraction is mostly a special kind of reverse-phase chromatography and can also be performed in a highly miniaturized scale (EP 1139087). US 2006198765 describes a solution that offers a 96-fold parallelized solid phase extraction or optionally also affinity chromatography for smallest volumes. In WO 02082051 parallelized affinity matrices are used for the pretreatment of samples for mass spectroscopy, too. This principle is applied in numerous other patent specifications.
Reverse-phase chromatography, solid phase extraction and affinity chromatography are based on the principle that the one or more analytes wash out of the accompanying substances and also possibly concentrated by this process, if required. But in said processes these methods always select special analytes in dependency of the characteristics of the analytes and the characteristics of the solid phase.
In the dialysis procedure described herein the accompanying substances and the solvent are removed without considerably influencing the composition of the essential analytes of the sample.
Dialysis procedures and the relevant arrangements are nowadays available for the desired volume range from about 10 μl up to several liters. Usually, dialysis tubes, mini sacks or cartridges are used. Here, the medium change is realized via the diffusion of the low-molecular matrix components through a semi-permeable membrane whereas the essential sample components are restrained from diffusing.
Semi-permeable membranes in the sense of this invention are membranes that do not allow particles, cells or molecules of a defined maximum size (cut-off) to pass, i.e. permeate. This is achieved by the pores that are provided in the membrane and mostly have a narrow size range. The membranes can be frits with pores of several 100 μm, filtration membranes with pores in the μm and sub-μm ranges and molecular screen membranes with a molecular weight ranging from a few hundred DA to several 100 kDa.
The diffusion is a process that is initiated by the Brownian movement according to the Fick's laws of diffusion (1).
                                          d            m                    =                      D            ⁢                                          q                ×                                  ⅆ                  c                                ×                                  ⅆ                  t                                                            ⅆ                l                                                    ,                            (        1        )            wherein:dm=quantity of the diffusing substanceD=diffusion coefficientq=diffusion cross-section or areadc=concentration difference, anddl=diffusion pathdt=diffusion time.
Additional factors that depend on the characteristics of the diffusing substance and of the membrane act at membranes.
For low-molecular matrix components the real diffusion times are directly proportional to the concentration gradient, the available area and inversely proportional to the diffusion path and normally amount to about 100 h for a diffusion path of 1.3 cm, about 1 h for 1.3 mm and about 0.3 sec for 13 μm (S. M. Rappoport, Medizinische Biochemie, Verl. Volk und Gesundheit, 1983, p. 39/40 [Medical Biochemistry, publishing company “Volk und Wissen”]).
Therefore, the diffusion will be useable for the efficient medium change, particularly for small volumes, if an appropriate geometry (large effective area, short diffusion paths) and suitable membranes (no interaction of the substances to be changed with the membrane) are used. Additionally, the concentration differences (dc in Formula 1) can be kept high by turbulences produced in the sample and/or the dialysis external liquid by appropriate mechanical systems and thus the diffusion through the membrane can be accelerated.
Bundles of hollow fibers with defined pores through which the sample liquid passes are also used for dialyses in the laboratory, and preferably for haemodialyses. In these methods, the same, mostly large-pored sample flows through a large number of bundled hollow fibers whereby the effective surface is considerably enlarged. Cartridges with semi-permeable membranes are also used for the medium change in the laboratory. Cartridges and hollow fibers have large total volumes due to the connection to pumps and require high pressures because the small cross-sections cause high flow resistances and the relatively thick walls act as long diffusion barriers. For this reason, it is difficult to integrate the two technical solutions in highly miniaturized or even parallelized systems.
In the laboratory, the dialysis can be performed in mini sacks or containers by simple pipette steps. Normally, the volumes of the samples are only changed insignificantly. If a suitable membrane type and pore size (molecular weight cut off) are selected, essential components of the biological samples will not be lost. However, a satisfying solution has not been developed so far for the simultaneous dialysis of a large number of small-volume samples that are of the same type but differ in the composition of essential analytes and matrix components. The “drop-dialysis”, i.e. dialysis with comparably macropored filtration membranes swimming on the dialysis, is designed for the quick desalting of minimum volumes down to few μl but it cannot be parallelized and does not deliver a satisfying quantitative result with respect to the sample amount to be re-obtained and the essential components contained in it.
Singular dialysis systems down to a minimum volume of presently 10 μl exist (e.g. U.S. Pat. No. 5,503,741) but the surface-volume-ratio is not optimal at all and a parallelization is not planned. Moreover, special microdialysis units have been developed for even smaller volumes for the diffusion-based in vivo extraction of low-molecular solutes (WO 2004032735) but nor for the medium change of essential sample components, Here, a maximum surface-volume-ratio is achieved in the small interior chamber partly formed by a semi-permeable membrane but in said chamber a liquid is transported that shall absorb diffusible substances from the outside existing live tissue sample and thus transfer them to an analysis process. A parallelization is excluded here.
U.S. Pat. No. 6,458,275 shows a possible way to perform the parallelized equilibrium dialysis to investigate binding constants in microplate screens. In this method an own external liquid is provided for each sample and, due to the low volume ratio of the external liquid/sample it cannot be used principally for an efficient medium change or for desalting.
Parallelized dialyses are performed in different solutions. It is possible to use up to 12 samples and a volume of minimally 20 μl (Pierce: Microdialyzer System) or up to 96 samples and a minimum volume of approximately 24 μl (preferably in microplate screens) (e.g. US 2004195163, US 2005019774, US 2005148066, SpectrumLabs: Spectra/PorMicrodialyzer). Due to the unfavorable surface-volume ratios, the relatively small diffusion areas and the resulting relatively long diffusion paths, a complete medium change takes many hours in this method. As a result of the evaporation capacity at the large surface into the enclosed air chamber, evaporation losses occur during this time even if the sample vessels are covered, and essential components of the samples can, change. For example, proteins can denature. Moreover, it is difficult to parallelize the samples from the microplate-well-shaped sample vessels with fragile membranes and to retrieve them without many losses. A standardization of the dialysis conditions for all samples is not feasible because of the contingency of the formation of the effective diffusion path that is caused by volume tolerances, meniscus formation, and hardly controllable air bubble inclusions and air cushion development.
Sample plates for the application in dialysis systems are known (DE 101 60 975 A1) and can be used to dialyze a large number of microsamples in the μl range. These vessels, the upper ends of which are open and the lower ends, i.e. the front surfaces of the sample vessels, are closed by a filtration membrane used for the drop-dialysis, have been arranged in a screen suitable for the liquid handling method of microplate technology. However, a further reduction of the dialysis time and a minimization of the risk of damage, particularly of the semi-permeable membrane during the loading phase (pipette movement), would be desirable. Furthermore, the dialysis time depends on the filling level (diffusion path dl in Formula 1) and consequently on the sample volume as well as on the pipette precision.
To sum up, a completely satisfying feasible, rapid, reproducible procedure in which a medium change can be unselectively performed without losses for essential components of samples in the volume range from <1 μl to 500 μl in a short period of time has not been introduced so far. Dialysis processes that get into the lower range of the mentioned volumes are not quantitative and cannot be parallelized; the methods that can be parallelized require larger volumes and have considerable disadvantages in terms of handling, the likely precision and the required dialysis times.
Concentration processes are principally possible by means of precipitation reactions, ultra-centrifugation, ultra-filtration, lyophylization, incl. the special form by SpeedVac, and adsorption methods.
Precipitations by means of neutral salts, acids and organic solvents are efficient methods to increase the concentration for many analytes, such as proteins. However, these procedures are not suitable for low-molecular substances and peptides, are also not “unselective” for many other analytes because the analyte and matrix properties define the precipitation efficiency, and moreover, they mostly denature a large number of proteins. In addition to this, it is difficult to miniaturize and parallelize precipitation reactions and normally the means used for precipitation have to be removed from the sample before the actual analysis.
Ultra-centrifugation and ultra-filtration cannot be used for μl volumes, require flux supporting means, such as centrifuges or vacuum, and it is difficult to perform them in a parallelized manner.
The kit recommended in US 2005133425 includes single elements for ultra-filtration for the purpose of a molecular-weight-selective concentration and thus for a selective enrichment of selected analytes.
The lyophylization, also by means of SpeedVac, is actually a method that can be well parallelized and miniaturized but it has the disadvantage that the concentration of all disturbing accompanying substances is also increased due to the applied physical principle to of solvent evaporation. If this method is continued until the total solvent removal, i.e. the dryness, it is combined with the risk that the essential analytes, particularly the proteins or special peptides, are not/or only partially soluble again.
Adsorptions by means of specific capture bonds are elegant methods to highly enrich analytes on surfaces, but they imply also a preselection of bound analytes. These procedures can be easily parallelized. Their application is described, for example, for numerous SELDI-supports in different designs. In WO 2005103718 they are described for glycoproteins, in US 2003027216 for immunoreactive analytes, in WO 2005070141 for different analytes to be selected, and in all these procedures, an up to 96-fold parallelization is intended.
But also unspecific adsorption processes, such as the above described reversed-phase chromatography in zip tips, select special, here particularly hydrophobic, analytes. Miniaturized chromatographic adsorptive methods (US 2003027216, WO 2005070141) mostly require a flux support, such as centrifugal acceleration or vacuum.
Apart from the adsorptive systems that are described above and are only useable to a limited extent, such as zip tips with the known disadvantages, an arrangement and a feasible method that allow medium changes in combination with a considerable concentration of numerous parallel samples in the μl range and without the selection of a part of the sample components do not exist.